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Identification, characterization, and application of a recombinant antigen for the serological diagnosis of feline hemotropic mycoplasma infections

Wolf-Jäckel, G A. Identification, characterization, and application of a recombinant antigen for the serological diagnosis of feline hemotropic mycoplasma infections. 2010, University of Zurich, Vetsuisse Faculty.

Abstract

In felids, three hemotropic mycoplasma species (hemoplasmas) have been described: Mycoplasma haemofelis (Mhf), ‘Candidatus Mycoplasma haemominutum’ (CMhm), and ‘Candidatus Mycoplasma turicensis’ (CMt). Particularly, Mhf may cause severe, potentially life-threatening hemolytic anemia. No routine serological assays for feline hemoplasma infections are available. Thus, the goal of this project was to identify an Mhf antigen (DnaK), to be used as a recombinant antigen in serological assays for the diagnosis of feline hemoplasma infections. The gene sequence of this protein was determined using consensus primers and blood samples from naturally and experimentally Mhf infected cats, and a naturally infected Iberian lynx (Lynx pardinus). Mhf DnaK was expressed recombinantly in an E. coli DnaK knock-out strain and purified using Ni-affinity, size exclusion, and anion exchange chromatography. It was then biochemically and functionally characterized and showed characteristics typical for DnaKs (secondary structure profile, thermal denaturation, ATPase activity, and DnaK complementation). Moreover, its immunogenicity was assessed using serum samples from experimentally hemoplasma infected cats. When the protein was applied in Western blot and enzyme-linked immunosorbent assays, it was recognized by sera from cats infected with Mhf, CMhm and CMt, respectively, but not from uninfected cats. This is the first description of a full-length purified recombinant feline hemoplasma antigen.

Abstract

In felids, three hemotropic mycoplasma species (hemoplasmas) have been described: Mycoplasma haemofelis (Mhf), ‘Candidatus Mycoplasma haemominutum’ (CMhm), and ‘Candidatus Mycoplasma turicensis’ (CMt). Particularly, Mhf may cause severe, potentially life-threatening hemolytic anemia. No routine serological assays for feline hemoplasma infections are available. Thus, the goal of this project was to identify an Mhf antigen (DnaK), to be used as a recombinant antigen in serological assays for the diagnosis of feline hemoplasma infections. The gene sequence of this protein was determined using consensus primers and blood samples from naturally and experimentally Mhf infected cats, and a naturally infected Iberian lynx (Lynx pardinus). Mhf DnaK was expressed recombinantly in an E. coli DnaK knock-out strain and purified using Ni-affinity, size exclusion, and anion exchange chromatography. It was then biochemically and functionally characterized and showed characteristics typical for DnaKs (secondary structure profile, thermal denaturation, ATPase activity, and DnaK complementation). Moreover, its immunogenicity was assessed using serum samples from experimentally hemoplasma infected cats. When the protein was applied in Western blot and enzyme-linked immunosorbent assays, it was recognized by sera from cats infected with Mhf, CMhm and CMt, respectively, but not from uninfected cats. This is the first description of a full-length purified recombinant feline hemoplasma antigen.

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